Hideaki Nakatsuji

Uric Acid Secretion from Adipose Tissue and Its Increase in Obesity

Background: Purine metabolism in adipose tissue is largely unknown. Results: Adipose tissue has abundant xanthine oxidoreductase activity. Uric acid is secreted from adipose tissues and cells, and the secretion is augmented in obese mice. Conclusion: Adipose tissue can secrete uric acid in mice. Significance: Dysfunction of obese adipose tissue could be related to overproduction of uric acid. Obesity is often accompanied by hyperuricemia. However, purine metabolism in various tissues, especially regarding uric acid production, has not been fully elucidated. Here we report, using mouse models, that adipose tissue could produce and secrete uric acid through xanthine oxidoreductase (XOR) and that the production was enhanced in obesity. Plasma uric acid was elevated in obese mice and attenuated by administration of the XOR inhibitor febuxostat. Adipose tissue was one of major organs that had abundant expression and activities of XOR, and adipose tissues in obese mice had higher XOR activities than those in control mice. 3T3-L1 and mouse primary mature adipocytes produced and secreted uric acid into culture medium. The secretion was inhibited by febuxostat in a dose-dependent manner or by gene knockdown of XOR. Surgical ischemia in adipose tissue increased local uric acid production and secretion via XOR, with a subsequent increase in circulating uric acid levels. Uric acid secretion from whole adipose tissue was increased in obese mice, and uric acid secretion from 3T3-L1 adipocytes was increased under hypoxia. Our results suggest that purine catabolism in adipose tissue could be enhanced in obesity.

The heart requires glycerol as an energy substrate through aquaporin 7, a glycerol facilitator

AIMS Cardiomyocytes require fatty acids and glucose for energy production. However, other nutrients and substrates that may serve as possible candidates for a cardiac energy source have not been fully studied. Several reports showed that a moderate expression of aquaporin 7 (AQP7), a member of the aquaglyceroporin family that is permeated by glycerol and water, is observed in heart tissue. However, the functional role of cardiac AQP7 is not clear. The aim of this study was to investigate the significance of glycerol as a cardiac energy substrate and to clarify the role of cardiac AQP7. METHODS AND RESULTS Heart function and morphology were examined in AQP7-knockout (KO) mice under basal conditions and during pressure overload [isoproterenol infusion and transverse aortic constriction (TAC)]. Glycerol uptake and glycerol-dependent ATP production were measured in AQP7-knockdown cardiac cells. Cardiac glycerol consumption was analysed in ex vivo beating hearts. Cardiac morphology and function in KO mice were similar to those of wild-type (WT) mice under basal conditions, although low glycerol and ATP content were noted in hearts of KO mice. In H9c2 cardiomyotubes, knockdown of AQP7 was associated with a significant reduction of glycerol uptake. The ex vivo heart study demonstrated that cardiac glycerol consumption levels in KO mice were significantly lower than those of WT mice. Furthermore, isoproterenol challenge induced severe left ventricular hypertrophy in KO mice, and TAC resulted in a higher mortality rate in KO mice than in WT mice. CONCLUSION The results indicate that AQP7 acts as a glycerol facilitator in cardiomyocytes and that glycerol is a substrate for cardiac energy production.

Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction.

AIMS Mineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects. METHODS AND RESULTS Corticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H(2)O(2), MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m+mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H(2)O(2), caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution. CONCLUSION Glucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS.

Reciprocal regulation of natriuretic peptide receptors by insulin in adipose cells

Atrial- and brain-type natriuretic peptides (ANP and BNP, respectively) have been shown to exert potent lipolytic action in adipocytes. A family of natriuretic peptide receptors (NPRs), NPR-1, NPR-2, and NPR-3, mediates their physiologic effects. NPR-1 and NPR-2 are receptor guanylyl cyclases, while NPR-3 lacks enzymatic activity and functions primarily as a clearance receptor for natriuretic peptides. ANP has a high affinity for NPR-1 and NPR-3 than other natriuretic peptides. There is a possibility that ANP may exhibit its lipolytic effect through the balance of NPR-1 and NPR-3 expressions in adipocytes. However, the regulation of adipose NPRs has not been fully elucidated. We here examined the regulation of mouse adipose NPRs by insulin, an anti-lipolytic hormone. Among the insulin target organs, NPR-1 mRNA levels were higher in white adipose tissue (WAT) than in liver and skeletal muscle. NPR-3 mRNA was expressed most abundantly in WAT. Fasting condition induced NPR-1 mRNA level while suppressed NPR-3 mRNA level in WAT. Administration of streptozotocin resulted in the increase of NPR-1 mRNA level while the decrease of NPR-3 mRNA level in WAT. In ob/ob mice, hyperinsulinemic model, NPR-1 mRNA level was lower whereas NPR-3 mRNA level was higher compared to lean control mice. In 3T3-L1 adipocytes, insulin significantly reduced NPR-1 mRNA level while increased NPR-3 mRNA levels both through phosphatidylinositol 3-kinase (PI3-kinase) pathway. In summary, NPR-1 and NPR-3 were highly expressed in WAT and adipose NPR-1 and NPR-3 were reciprocally regulated by insulin. This study suggests that insulin may efficiently promote lipogenesis partly by reducing the lipolytic action of ANP through the opposite regulation of NPR-1 and NPR-3.

High circulating levels of S100A8/A9 complex (calprotectin) in male Japanese with abdominal adiposity and dysregulated expression of S100A8 and S100A9 in adipose tissues of obese mice

S100A8/A9 complex, calprotectin, which serves as an endogenous ligand for immune pathways, is associated with atherosclerosis. These proteins are reported to have several functions such as activating NADPH oxidase, binding toll-like receptor 4 and associated with the receptor for advanced glycation end-products. We recently reported S100A8 mRNA was highly expressed in mouse white adipose tissues and differentiated 3T3-L1 adipocytes. However, regulation of S100A9 expression in murine adipose tissue remains to be elucidated. The results of our studies in male Japanese, obese and control mice and cultured cells showed: (1) serum levels of S100A8/A9 complex, calprotectin, correlated with visceral fat area, body mass index, subcutaneous fat area, and leukocyte count in 500 Japanese men, and (2) higher mRNA expression levels of S100A8 in mature adipocyte fraction and S100A9 in stromal vascular cell fraction of obese mice, compared with those of lean mice. Overexpression of S100A8 and S100A9 in obese adipose tissue may be involved, at least partly, in not only high circulating levels of S100A8/A9 complex in abdominal obesity but also adipose and systemic tissue inflammation.

Dynamic Changes of Adiponectin and S100A8 Levels by the Selective Peroxisome Proliferator–Activated Receptor-γ Agonist Rivoglitazone

Objective—Accumulating evidence indicates that the regimen to increase adiponectin will provide a novel therapeutic strategy for metabolic syndrome. Here, we tested the effect of a potent and selective peroxisome proliferator–activated receptor-&ggr; agonist, rivoglitazone (Rivo), a newly synthesized thiazolidinedione derivative, on adiponectin, insulin resistance, and atherosclerosis. Methods and Results—ob/ob mice, apolipoprotein E knockout (apoE KO) mice, and apoE and adiponectin double knockout mice were administered pioglitazone, Rivo, or no compound. Remarkable elevation of plasma adiponectin was observed, especially in Rivo-treated ob/ob mice. Rivo ameliorated insulin resistance in ob/ob mice and reduced atherosclerotic areas in apoE KO mice compared with the pioglitazone group but failed to decrease atherosclerotic areas in double knockout mice. Among adipose mRNAs, adipose S100A8, which activates Toll-like receptor 4–dependent signal transduction cascades and locates upstream of inflammation, was markedly increased in ob/ob mice, and its increase was completely reversed by Rivo treatment. In RAW264.7 macrophage cells and 3T3-L1 adipocytes, Rivo significantly reduced S100A8 mRNA levels. Conclusion—The peroxisome proliferator–activated receptor-&ggr; agonist Rivo remarkably enhanced adiponectin in plasma and decreased adipose S100A8 mRNA levels in obese mice. Rivo treatment apparently ameliorated insulin resistance in ob/ob mice and reduced atherosclerosis in apoE KO mice, partly through adiponectin.

Relationship between visceral fat accumulation and urinary albumin-creatinine ratio in middle-aged Japanese men

OBJECTIVE Chronic kidney disease including microalbuminuria relates to cardiovascular disease (CVD). Microalbuminuria is also known to be a marker of generalized endothelial dysfunction. The metabolic syndrome which encompasses visceral fat accumulation and various metabolic disorders, has also an increase in albuminuria and relates to CVD. However, the relationship between visceral fat accumulation and albuminuria remains to be defined. The present study investigated the relationship between visceral fat accumulation and urinary albumin-creatinine ratio (UACR) in Japanese men. METHODS This study group comprised 1990 Japanese male subjects, who were employees of a city office, had undergone annual health check-up. Urinary albumin was collected from a single spot urine specimen collected anytime between morning and afternoon. Visceral fat area was estimated (eVFA) by the bioelectrical impedance analysis method. RESULTS Log-UACR correlated with age, log-body mass index (BMI), log-waist circumference (WC), log-eVFA, log-adiponectin, blood pressure, serum lipids and hemoglobin A1c (HbA1c). Stepwise multiple regression analysis identified log-eVFA, as well as HbA1c, blood pressure, log-TG, and age, as a significant determinant of log-UACR. Moreover, subjects with eVFA > or = 100 cm(2) had significantly higher UACR than those with eVFA <100 cm(2), irrespective of BMI. UACR was significantly worse in subjects with high numbers of metabolic risk factors, and moreover in subjects with eVFA > or = 100 cm(2) than in those with eVFA <100 cm(2). CONCLUSION These results suggested that visceral fat accumulation is associated with an increase in UACR. Evaluation of both visceral fat accumulation and urinary albumin may be important for preventing atherosclerotic diseases.

Binding of adiponectin and C1q in human serum, and clinical significance of the measurement of C1q-adiponectin / total adiponectin ratio

OBJECTIVE Adiponectin and C1q have similar sequences, exist abundantly in blood, and are produced by adipose tissues. The aim of this study was to examine whether adiponectin and C1q form protein-complex in blood and to know the clinical significance of the C1q-adiponectin (C1q-APN) complex in serum. METHODS The direct interaction between adiponectin and C1q was investigated by far western blotting and co-immunoprecipitation. The relationship between serum C1q-APN and various clinical features was analyzed in 329 Japanese men who underwent health check-up, including measurements of visceral (VFA) and subcutaneous fat area (SFA) by computed tomography (Victor-J study). RESULTS Adiponectin bound to C1q in vitro and C1q-APN complex existed in human blood. C1q-APN complexes were identified in high- and middle-molecular weight forms of adiponectin in human serum by gel-filtration chromatography. Stepwise multiple regression analysis identified body mass index, VFA and SFA as significant determinants of serum C1q-APN level. Serum C1q-APN/Total-APN ratio correlated positively with cardiovascular risk factor accumulation in subjects with VFA ≥100 cm(2). CONCLUSIONS These results indicate that high- and middle-molecular forms of adiponectin partly consist of adiponectin-complex with other proteins including C1q and that the blood C1q-APN/Total-APN ratio may serve as a biomarker of the metabolic syndrome in general male subjects.

Adiponectin Protein Exists in Aortic Endothelial Cells

Aims Inflammation is closely associated with the development of atherosclerosis and metabolic syndrome. Adiponectin, an adipose-derived secretory protein, possesses an anti-atherosclerotic property. The present study was undertaken to elucidate the presence and significance of adiponectin in vasculature. Methods and Results Immunofluorescence staining was performed in aorta of wild-type (WT) mice and demonstrated that adiponectin was co-stained with CD31. Thoracic aorta was cut through and then aortic intima was carefully shaved from aorta. Western blotting showed the existence of adiponectin protein in aortic intima, while there was no adiponectin mRNA expression. Adiponectin knockout (Adipo-KO) and WT mice were administered with a low-dose and short-term lipopolysaccharide (LPS) (1 mg/kg of LPS for 4 hours). The endothelium vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were highly increased in Adipo-KO mice compared to WT mice after LPS administration. Conclusions Adiponectin protein exists in aortic endothelium under steady state and may protect vasculature from the initiation of atherosclerosis.

Visualized macrophage dynamics and significance of S100A8 in obese fat

Significance Infiltration of immune cells into adipose tissue has been observed in obesity, but, until now, these data were based on immunohistological microscopic analysis. Here, to our knowledge for the first time, we applied an intravital multiphoton imaging technique to adipose tissue with lysozyme M-EGFP transgenic (LysMEGFP) mice whose EGFP was expressed in the myelomonocytic lineage. Mobility of LysMEGFP-positive macrophages was activated just 5 d after high-fat and high-sucrose (HF/HS) diet before the development of obesity. Furthermore, a significant increase of S100A8, one of the alarmins, was detected in fat tissue just 5 d after HF/HS diet. S100A8 stimulated chemotactic migration, and neutralization of S100A8 suppressed the HF/HS diet-induced activation of LysMEGFP-positive cells. Time-lapse intravital imaging first identified the very early event exhibiting increased mobility of adipose macrophages. Chronic low-grade inflammation of adipose tissue plays a crucial role in the pathophysiology of obesity. Immunohistological microscopic analysis in obese fat tissue has demonstrated the infiltration of several immune cells such as macrophages, but dynamics of immune cells have not been fully elucidated and clarified. Here, by using intravital multiphoton imaging technique, to our knowledge for the first time, we analyzed and visualized the inflammatory processes in adipose tissue under high-fat and high-sucrose (HF/HS) diet with lysozyme M-EGFP transgenic (LysMEGFP) mice whose EGFP was specifically expressed in the myelomonocytic lineage. Mobility of LysMEGFP-positive macrophages was shown to be activated just 5 d after HF/HS diet, when the distinct hypertrophy of adipocytes and the accumulation of macrophages still have not become prominent. Significant increase of S100A8 was detected in mature adipocyte fraction just 5 d after HF/HS diet. Recombinant S100A8 protein stimulated chemotactic migration in vitro and in vivo, as well as induced proinflammatory molecules, both macrophages and adipocytes, such as TNF-α and chemokine (C-C motif) ligand 2. Finally, an antibody against S100A8 efficiently suppressed the HF/HS diet-induced initial inflammatory change, i.e., increased mobilization of adipose LysMEGFP-positive macrophages, and ameliorated HF/HS diet-induced insulin resistance. In conclusion, time-lapse intravital multiphoton imaging of adipose tissues identified the very early event exhibiting increased mobility of macrophages, which may be triggered by increased expression of adipose S100A8 and results in progression of chronic inflammation in situ.